Splice isoform-specific RNAi as therapy for Spinocerebellar Ataxia type 6

描述(申请人提供)：脊髓小脑性共济失调-6型(SCA6)是一种主要由编码CAG重复序列的多谷氨酰胺(PolyQ)引起的遗传性共济失调，该重复序列位于Cav2.1钙通道基因CACNA1a的3‘端附近。选择性小脑浦肯野神经元的丢失是SCA6的病理特征，SCA6是一种进展缓慢、身体虚弱的疾病，目前尚无治疗方法。这里提出的临床前研究将测试一种新的剪接-异构体特异性RNA干扰(SIS-RNAi)治疗SCA6的方法，使用该疾病的遗传准确的小鼠模型。Cav2.1转录本3‘端的选择性剪接产生了编码两类Cav2.1变体的mRNA异构体，一种缺失，另一种在蛋白质的c-末端附近含有多Q结构域。在SCA6中，最近的研究表明，编码突变的(扩展的)包含多Q的Cav2.1变体的剪接异构体水平异常增加。含有毒性多Q的Cav2.1变异体的水平似乎只在小脑浦肯野神经元中增加，这可能解释了它们在SCA6中的选择性退化。总体假设是，我们的研究小组开发的一种新的SIS-RNAi方法将选择性地抑制浦肯野神经元中含有多聚Q的Cav2.1变体的表达，因此将被证明对SCA6具有显著的治疗益处。目的1建立重组腺相关(RAAV)介导的SIS-RNAi分子选择性抑制人多聚Q编码Cav2.1剪接异构体在SCA6敲入小鼠模型浦肯野神经元中表达的能力。SCA6基因敲除小鼠在内源性Cav2.1启动子的控制下表达扩增的多Q编码Cav2.1转录本。重要的是，这些小鼠忠实地概括了在人类SCA6中观察到的运动和组织病理学特征。目的2研究长期表达rAAV-SIS-RNAi对SCA6敲门小鼠进行性神经功能障碍的分子和行为学影响。拟议研究的主要影响是，他们有望提供临床前数据，支持SIS-RNAi作为人类SCA6治疗的发展。此外，他们还将突出SIS-RNAi作为其他遗传性疾病治疗策略的潜力，并打造有助于我们理解Cav2.1通道功能和调节的新试剂。

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type-6 (SCA6) is a dominantly inherited ataxia caused by a polyglutamine (polyQ)- encoding CAG repeat expansion near the 3'-end of the Cav2.1 calcium channel gene, CACNA1a. Selective loss of cerebellar Purkinje neurons is the pathological hallmark of SCA6, a slowly progressive and debilitating disease for which there is no treatment. The preclinical studies proposed here will test a novel splice-isoform- specific RNA interference (SIS-RNAi) therapy approach for SCA6, using a genetically accurate mouse model of the disease. Alternative splicing at the 3'-end of the Cav2.1 transcript produces mRNA isoforms that encode two classes of Cav2.1 variants, one lacking and one containing the polyQ domain near the c-terminal end of the protein. In SCA6, recent studies have shown an aberrant increase in the levels of splice isoforms encoding for mutant (expanded) polyQ-containing Cav2.1 variants. Levels of toxic polyQ-containing Cav2.1 variants appear to increase only in cerebellar Purkinje neurons, potentially explaining their selective degeneration in SCA6. The overall hypothesis is that a novel SIS-RNAi approach developed by our research group will selectively suppress expression of polyQ-containing Cav2.1 variants in Purkinje neurons and thus will prove to be of significant therapeutic benefit in SCA6. Aim 1 will establish the capacity of recombinant adeno-associated (rAAV)-delivered SIS-RNAi molecules to selectively suppress the expression of human, polyQ-encoding Cav2.1 splice-isoforms in Purkinje neurons of an SCA6 knock-in mouse model. SCA6 knock-in mice express expanded polyQ-encoding Cav2.1 transcripts under the control of the endogenous mouse Cav2.1 promoter. Importantly, these mice faithfully recapitulate motor and histo-pathological features observed in human SCA6. Aim 2 will examine the molecular and behavioral effects of long-term rAAV-SIS-RNAi expression on the progressive neurological dysfunction observed in SCA6 knockin mice. The primary impact of the proposed studies is that they are expected to provide preclinical data supporting the development of SIS-RNAi as therapy for humans with SCA6. Moreover, they will highlight the potential of SIS-RNAi as a therapeutic strategy in other hereditary diseases and forge novel reagents useful to our understanding of Cav2.1 channel function and regulation.